发酵白芥子穴位敷贴对支气管哮喘大鼠气道炎症和Th17/Treg免疫失衡的影响

Effect of acupoint application of fermented white mustard seed on airway inflammation and Th17/Treg immune imbalance in rats with bronchial asthma

目的 探究发酵白芥子穴位敷贴对支气管哮喘大鼠气道炎症和辅助性T细胞17(Th17)/调节性T细胞(Treg)免疫失衡的影响。方法 将48只SPF级SD大鼠随机分为空白组、模型组、地塞米松组、发酵组、非发酵组及假贴组,每组8只。空白组用生理盐水代替干预,其余组大鼠均用卵清蛋白和氢氧化铝混合液致敏建立支气管哮喘模型。造模成功后,每天雾化激发1 h前,地塞米松组给予腹腔注射地塞米松1 mg/kg,1次/d,共10次;发酵组、非发酵组将药物贴敷于大鼠大椎、膻中、肺俞(双侧)、脾俞(双侧)、肾俞(双侧)穴,每次5 h,1次/d,共10次;假贴组用同样方法贴敷凡士林;空白组和模型组不做任何处理。于末次雾化激发时观察各组大鼠哮喘行为并计分;干预结束后,取肺组织行HE染色观察其病理形态,取大鼠血清和肺泡灌洗液,ELISA法检测Th17、Treg相关细胞因子白细胞介素-17(IL-17)、白细胞介素-22(IL-22)、白细胞介素-10(IL-10)和白细胞介素-35(IL-35)水平,Western blot法检测肺组织中IL-17、IL-22、IL-10、IL-35蛋白表达情况。结果 与空白组比较,模型组、假贴组大鼠支气管黏膜上皮细胞受损脱落,管腔明显狭窄,气道壁不完整,肺泡内炎症细胞浸润明显,肺泡间隔明显增厚;哮喘行为学评分、血清及肺泡灌洗液中IL-17和IL-22水平、肺组织中IL-17和IL-22蛋白相对表达量均明显升高(P均<0.05),血清及肺泡灌洗液中IL-10和IL-35水平、肺组织中IL-10和IL-35蛋白相对表达量均明显降低(P均<0.05)。与模型组和假贴组比较,地塞米松组、发酵组、非发酵组大鼠支气管上皮较完整,偶见上皮细胞脱落,支气管黏膜偶见炎性细胞浸润,肺间质轻微增生;哮喘行为学评分、血清及肺泡灌洗液中IL-17和IL-22水平、肺组织中IL-17和IL-22蛋白相对表达量均明显降低(P均<0.05),血清及肺泡灌洗液中IL-10和IL-35水平、肺组织中IL-10和IL-35蛋白相对表达量均明显升高(P均<0.05);地塞米松组、发酵组、非发酵组间肺组织病理形态、血清及肺泡灌洗液中各炎症指标水平和肺组织中蛋白表达量比较差异均无统计学意义(P均>0.05)。结论 发酵白芥子穴位敷贴能有效减轻支气管哮喘大鼠哮喘发作症状和气道炎症,降低体内炎性细胞因子水平,调节Th17/Treg免疫失衡状态。

Objective It is to explore the effect of acupoint application of fermented white mustard seed on airway inflammation and Th17/Treg immune imbalance in rats with bronchial asthma.Methods Forty-eight SPF SD rats were randomly divided into blank group,model group,dexamethasone group,fermentation group,non-fermentation group and sham paste group,with 8 rats in each group.The rats in the blank group were treated with normal saline instead of intervention,and the rats in other groups were sensitized with the mixture of ovalbumin and aluminum hydroxide to establish models of bronchial asthma.After successful modeling,the dexamethasone group was given dexamethasone 1 mg/kg by intraperitoneal injection at 1 hour before nebulization stimulation,once daily,totally for 10 times;the fermentation group and non-fermentation group was treated with the drug by acupoint application at acupoints of Dazhui,Danzhong,Feishu(bilateral),Pishu(bilateral) and Shenshu(bilateral),5 hours per time,once daily,total for 10 times;the sham paste group was treated with vaseline by the same method;the blank group and model group were treated with no treatment.The asthma behavior of rats in each group was observed and scored at the last aerosol challenge;at the end of treatment,the lung tissue was taken to observe the pathological changes by HE staining;the bronchoalveolar lavage fluid(BALF) and serum were taken to detect the levels of Th17,Treg-related cytokines interleukin(IL)-17,IL-22,IL-10 and IL-35 by ELISA method,and the expressions of IL-17,IL-22,IL-10 and IL-35 proteins in lung tissue were detected by Western blotting.Results Compared with the blank group,the bronchial mucosal epithelial cells were impaired,with obvious lumen stenosis,incomplete airway wall,intraalveolar inflammatory cell infiltration,and alveolar septa in the model group and sham paste group;the asthma behavioral score,the levels of IL-17 and IL-22 in serum and BALF,the relative expressions of IL-17 and IL-22 protein in lung tissue were significantly increased(all P<0.05),the levels of IL-10 and IL-35 in serum and BALF,and the relative expressions of IL-10 and IL-35 in lung tissue were significantly decreased(all P<0.05).Compared with the model group and sham paste group,the bronchial epithelium was complete,with isolated epithelial cells,inflammatory cell infiltration of bronchial mucosa,and mild pulmonary hyperplasia in the dexamethasone group,fermentation group and non-fermentation group;the asthma behavioral score,the levels of IL-17 and IL-22 in serum and BALF,the relative expressions of IL-17 and IL-22 protein in lung tissue were significantly decreased(all P<0.05),the levels of IL-10 and IL-35 in serum and BALF,and the relative expressions of IL-10 and IL-35 in lung tissue were significantly increased(all P<0.05);There were no statistically significant differences in pathological morphology of lung histology,the levels of various inflammatory indexes in serum and BALF,and protein expression in lung tissues among the dexamethasone group,the fermentation group,and non-fermentation group(P all >0.05).Conclusion Acupoint application of fermented white mustard seed can effectively relieve asthma symptoms and tracheitis,reduce the level of inflammatory cytokines and regulate the immune imbalance of Th17/Treg in asthmatic rats.